1. Field of the Invention
This invention pertains to a method of separating free from bound fractions in an immunoassay procedure and to a novel immunochemical composite for use therein.
2. Description of the Prior Art
The end-point of any competitive binding analysis involves determining the relative proportion of antigen (or hapten) that is free and antigen (or hapten) that is bound to the saturable binder. Current basic separation techniques involve differential migration of bound and free fractions (i.e., paper chromatoelectrophoresis, gel filtration), adsorption methods (i.e., charcoal, silicates), fractional precipitation (i.e., ammonium sulfate, polyethylene glycol), and a double antibody method. According to J. G. Ratcliffe, Br. Med. Bull., 30:32 (1974), an ideal separation technique should fulfill the following criteria:
A. It must completely separate bound and free fractions with a wide margin for error in the conditions used for separation.
B. It must be simple, quick, cheap, and use reagents and equipment that are readily available.
C. It should be unaffected by plasma or serum. Ratcliffe further notes that for general clinical application, all manipulations should be performed in a single tube, be suitable for automation, and be applicable to a wide range of antigens or haptens (i.e., small peptides and steroids as well as large molecular weight proteins).
Although the double antibody method for separation of free and bound fractions in radioimmunoassay (RIA) systems in currently one of the most widely employed separation techniques and under optimum conditions satisfies most of the criteria mentioned above, it does possess certain inherent disadvantages. See J. G. Ratcliffe, supra; K. Seki and M. Seki, Endocrinol. Japan, 20:121 (1973); and P. Koninckx, R. Bouillon, and P. De Moor, Acta Endocrinol., 81:43 (1976). Since carrier protein must be added, large quantities of selected precipitating antibody are required and thus the method is expensive. It requires considerable length of time for the immunoprecipitation reaction to reach equilibrium (24 to 48 hours at 4.degree. C.). Finally, due to the possibility of aspecific interferences by factors present in the serum, the conditions of the assay must be meticulously evaluated before establishing an assay system.
Antibodies coupled to cyanogen bromide-activated dextran and cellulose particles are widely used in RIA systems as a result of the work of Wide, Porath, and Axen (L. Wide and J. Porath, Biochem. Biophys. Acta, 130:257 (1966)), R. Axen, J. Porath and S. Ernback, Nature (Lond.), 214:1302 (1967), and L. Wide, Acta Endocrinol. (Copenhagen) Suppl. No. 142:207 (1969)). A. E. Bolton and W. H. Hunter, Biochemica et Biophysica Acta, 329:318 (1973), reported that recovery of primary antibody activity tended to be higher on cyanogen bromde activated solid preparations of antisera to haptens and small peptides than to similar solid phase preparations of antisera to large molecular weight protein hormones. Thus one of the main disadvantages in the use of solid phase primary antibodies in RIA systems is that as a result of the loss of antibody titer and avidity which often occurs during the coupling step, one must develop longer, more expensive and more complicated systems (i.e., sequential assays). (A. Zeltner and P. E. Duly, Clin. Chem., 20:5 (1974)). Recently, F. C. Den Hollander, A. H. W. M. Shurrs, and H. Zan Hell, J. Immunol. Methods, 1:247 (1972), developed a new separation method employing second antiserum coupled to an insoluble matrix by use of cyanogen bromide and called the separation method and double antibody solid phase (DASP) method. Although the titer and avidity of the second antibody is most certainly reduced in these solid preparations, the primary antibody reaction remains unaltered and thus one of the main disadvantages of solid phase systems is circumvented. In fact, the DASP method possesses the following advantages over the more conventional soluble double antibody method of separation of free and bound fractions:
A. Since solid preparations of precipitating antibody require little or no carrier protein, less second antibody is required to precipitate the first antibody.
B. The DASP method requires less time for complete separation of free and bound fractions.
C. Aspecific interferences by factors present in the serum are totally absent with the DASP precipitation if one works in the area of excess second antibody. In fact, once optimal conditions for precipitation of the immune complex are established, no frequency reassessment is required.
For all the pluses one obtains with solid preparations of precipitating antibody, there remains one mechanical disadvantage common to all solid phase assays. It is necessary in all solid phase assays to agitate the reactants continuously with the additional extra task of first stoppering the assay and then centrifuging and unstoppering them prior to the washing step. V. Chan, C. Merrett, J. Landon, A. M. Linden, and M. Joustra, Ann. Clin. Biochem., 12:173 (1975), coupled primary antibodies to cyanogen bromide activated Sephadex G25 brand, ultrafine, bead-formed, dextran gel (less than 10 .mu. particle size) and reported that will small incubation volumes (300 to 400 microliters) it is possible for the antibody reaction to proceed without the need for vertical rotation or any other means of continuous agitation.
It has been discovered that immunochemical composites containing a finely divided derivatized polysaccharide matrix wherein the polysaccharide matrix has an average wet maximum dimension of 1 to 18 .mu. and also containing positively charged imidoesters which covalently couple said finely divided, derivatized, polysaccharide matrix to an antibody are excellent means for separating free from bound fractions without the need for vertical rotation or any other type of continued agitation. Further, the novel immunochemical composites within the scope of this invention display an amount of activity which far exceeds the activity displayed by the immunochemical composites prepared by Chan et al.